Within a nerve terminal, synaptic vesicles exclusively fuse at the active zone. The active zone consists of a protein scaffold that is anchored to the plasma membrane and forms release sites precisely opposed to postsynaptic receptors. Interactions between active zone proteins and Ca2+ channels have long been of central interest. Ca2+ influx through channels of the CaV2 family triggers release, and their exact positioning supports the sub-millisecond timing of synaptic transmission and determines synaptic strength. There are two competing models for roles and mechanisms of Ca2+ channels in synapse and active zone assembly. First, Ca2+ channels may be essential for synapse structure. Second, the active zone may recruit Ca2+ channels to release sites, implying that synapse structure is CaV2 independent. It has been difficult to distinguish between these models because the complexity of the Ca2+ channel gene family and their auxiliary subunits leads to extensive redundancy. Furthermore, precisely localizing Ca2+ channels has been challenging. We have overcome these hurdles by generating conditional triple knockout mice to remove all pore-forming a1 subunits of CaV2 channels, and by adapting superresolution microscopy to assess Ca2+ channel localization. Our data confirm that Ca2+ flux through these channels is essential for release triggering. Based on our preliminary data, we hypothesize that active zone assembly is independent of CaV2 channels, but instead the active zone targets CaV2 channels with nanoscale precision to release sites. Our experimental plan tests this hypothesis from three independent angles and dissects underlying mechanisms. In aim 1, we assess the competing models by removing the pore forming a1 subunits, followed by assessment of synapse and active zone structure and function. We then propose rescue experiments to assess which sequences of CaV2 channels are required for their targeting, and we test which CaV2 sequences are sufficient to confer active zone targeting onto non-CaV2 channels. In aim 2, we determine the precise presynaptic localization of auxiliary subunits and assess whether their presynaptic targeting depends on a1. We then test whether functional roles of these auxiliary subunits require the presence of a1. In aim 3, we address molecular mechanisms for CaV2 targeting from the perspective of active zone scaffolds. We first determine the order of arrival of active zone and CaV2 proteins during active zone assembly, and we then determine localization and function of CaV2s and their subunits in mutants that lack specific active zone proteins. This grant will test two fundamentally different models of the relationship between Ca2+ channels and the active zone, and dissects the mechanisms that underlie Ca2+ channel anchoring at the target membrane. Precise understanding of these mechanisms is important for understanding synapses in health and disease.